Chimeric antigen receptors with an optimized hinge region

ABSTRACT

The present invention relates to multi-functional proteins which comprise (i) a signal peptide, (ii) a target specific recognition domain, (iii) a linker region, connecting domain (ii) and domain (iv) which comprises a specific modified hinge region of the human CD8 alpha-chain, and (iv) an effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors with an optimized linker or hinge region that are suitable for generating target-specific effector cells, for use as a medicament, in particular in the treatment of cancer and in adoptive, target-cell specific immunotherapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.13/821,491, filed May 28, 2013 which is now U.S. Pat. No. 9,212,229;which is a National Stage application of International ApplicationNumber PCT/EP2011/004490, filed Sep. 6, 2011; which claims priority toEuropean Patent Application No. 10009345.9, filed Sep. 8, 2010; all ofwhich are incorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled“SeqList-14May13-ST25.txt”, which was created on May 14, 2013, and is 25KB. The entire content is incorporated herein by reference in itsentirety.

The present invention relates to multi-functional proteins whichcomprise (i) a signal peptide, (ii) a target specific recognitiondomain, (iii) a linker region, connecting domain (ii) and domain (iv)which comprises a specific modified hinge region of the human CD8alpha-chain, and (iv) an effector domain. The present inventionfurthermore relates to nucleic acids encoding the proteins, expressionconstructs for expressing the protein in a host cell and host cells. Theproteins of the invention are chimeric antigen receptors with anoptimized linker or hinge region that are suitable for generatingtarget-specific effector cells, for use as a medicament, in particularin the treatment of cancer and in adoptive, target-cell specificimmunotherapy.

BACKGROUND OF THE INVENTION

T lymphocytes recognize specific antigens through interaction of the Tcell receptor (TCR) with short peptides presented by majorhistocompatibility complex (MHC) class I or II molecules. For initialactivation and clonal expansion, naïve T cells are dependent onprofessional antigen-presenting cells (APCs) that provide additionalco-stimulatory signals. TCR activation in the absence of co-stimulationcan result in unresponsiveness and clonal anergy. To bypassimmunization, different approaches for the derivation of cytotoxiceffector cells with grafted recognition specificity have been developed.Chimeric antigen receptors (CARs) have been constructed that consist ofbinding domains derived from natural ligands or antibodies specific forcell-surface antigens, genetically fused to effector molecules such asthe TCR alpha and beta chains, or components of the TCR-associated CD3complex. Upon antigen binding, such chimeric antigen receptors link toendogenous signaling pathways in the effector cell and generateactivating signals similar to those initiated by the TCR complex. Sincethe first reports on chimeric antigen receptors, this concept hassteadily been refined and the molecular design of chimeric receptors hasbeen optimized (for a review see Uherek et al., 2001). Aided by advancesin recombinant antibody technology, chimeric antigen receptors targetedto a wide variety of antigens on the surface of cancer cells and ofcells infected by human immunodeficiency virus (HIV) have been generated(for a review see Uherek et al., 2001).

US 2007/0031438 A1 describes a CAR comprising a binding domain of anantibody against prostate specific membrane antigen (PSMA), a modifiedCD8 hinge in which at least one of the cysteine residues has beenmutated and a zeta signaling domain of the T cell receptor. Inparticular, US 2007/0031438 A1 uses a human CD8 hinge region with aminoacid positions 135 to 180 (according to the amino acid numbering ofSwissprot P01732), wherein the cysteine in position 164 is substitutedwith alanine.

Fitzer-Attas et al. (1998) describe a CAR comprising a non modified CD8hinge region with amino acid positions 116 to 208 (according to theamino acid numbering of Swissprot P01732), which comprises threecysteine residues at positions 164, 181 and 206. The chimeric receptorfurthermore uses kinase domains as effector domain.

WO 2008/045437 A2 describes CARs comprising as an extracellular bindingportion, a single chain antibody portion that binds to EGFRvIII, atransmembrane portion derived from human CD8 alpha or CD28, and anintracellular signaling portion derived from human CD3 zeta. Inparticular, WO 2008/045437 A2 describes chimeric T cell receptorproteins with a non modified CD8 hinge region with amino acid positions135 to 205, 135 to 203 or 135 to 182 (according to the amino acidnumbering of Swissprot P01732), each comprising cysteine residues inpositions 164 and 181.

WO 95/30014 A1 describes a CAR comprising an antigen binding domainderivable from a monoclonal antibody directed against a suitable antigenon a tumor cell (such as scFv(FRP5)), a hinge region comprising from 40to 200 amino acids and a functional zeta chain derivable from the T cellantigen receptor. In particular, the CAR of WO 95/30014 A1 uses the nonmodified murine CD8 hinge region with amino acid positions 132 to 191(according to the amino acid numbering of Swissprot P01731), comprisinga cysteine residue in position 178.

US 2008/0260738 A1 describes antibody fusion proteins comprising atleast two Fc monomers and at least one linker, wherein a modified CD8hinge region is used for linking the two Fc monomers. In particular, US2008/0260738 A1 uses modified CD8 hinge regions with amino acidpositions 131 to 170 or 136 to 169 (according to the amino acidnumbering of Swissprot P01732), wherein the cysteine in position 164 issubstituted with serine.

The present invention aims to provide optimized chimeric antigenreceptors which allow more efficient surface expression and highfunctionality in lymphocytes.

It is a further objective of the present invention to provide means andmethods for generating antigen-specific effector cells as well as meansand methods for the use in adoptive, target-cell specific immunotherapyand for treatment of cancer.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by amulti-functional or multi-domain protein comprising

(i) a signal peptide;

(ii) a target specific recognition domain;

(iii) a linker region, connecting domain (ii) and domain (iv); and

(iv) an effector domain.

According to the invention, the linker region (iii) is a modified hingeregion of the human CD8 alpha-chain, wherein the human CD8 alpha-chainhinge region is modified by replacing the cysteine residue(s) with (a)serine residue(s) or deleting the cysteine residue(s); and wherein theamino acid sequence of the modified hinge region of the human CD8alpha-chain has at least 95% sequence identity to the amino acidsequence of SEQ ID NO. 2 or wherein the linker region (iii) has theamino acid sequence of SEQ ID NO. 2.

According to the present invention this object is furthermore solved bya nucleic acid encoding the multi-functional protein.

According to the present invention this object is furthermore solved byan expression construct for expressing the multi-functional protein.

According to the present invention this object is furthermore solved bya host cell expressing the multi-functional protein or comprising thenucleic acid or the expression construct.

According to the present invention this object is furthermore solved byusing the multi-functional protein, nucleic acid, or expressionconstruct for generating antigen-specific effector cells.

According to the present invention this object is furthermore solved bythe multi-functional protein, nucleic acid, expression construct or hostcell for use as a medicament.

According to the present invention this object is furthermore solved bythe multi-functional protein, nucleic acid, expression construct or hostcell for use in the treatment of cancer.

According to the present invention this object is furthermore solved bythe multi-functional protein, nucleic acid, expression construct or hostcell for use in adoptive, target-cell specific immunotherapy.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is tobe understood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. For the purpose of thepresent invention, all references cited herein are incorporated byreference in their entireties.

Multi-Functional, Multi-Domain Proteins

As described above, the present invention provides multi-functionalproteins comprising several domains, namely

(i) a signal peptide;

(ii) a target specific recognition domain;

(iii) a linker region, connecting domain (ii) and domain (iv); and

(iv) an effector domain.

The multi-functional proteins of the invention are chimeric antigenreceptors characterized by an optimized hinge region (linker region).

The proteins of the invention are preferably cell surface receptorproteins and, thus, comprise an extracellular portion (domains (i) and(ii) and (iii)), a transmembrane portion (contributed by/comprised indomain (iv)) and a cytoplasmic portion (contributed by/comprised indomain (iv)), and can thus be inserted into the plasma membrane of thehost cell. The functionality of the proteins of the invention within ahost cell is detectable in an assay suitable for demonstrating thesignaling potential of said protein upon binding of a particular ligand.Such assays are available to the skilled artisan.

Upon binding to the target, such chimeric antigen receptors link toendogenous signaling pathways in a cell (an effector cell) and generatecertain activating signals (depending on the effector domain).

The expression of chimeric antigen receptors (CAR) with defined targetspecificity (such as target-cell specificity) in lymphocytes and othereffector cells of the immune system (such as T cells or natural killer(NK) cells) results in genetically modified variants of said cells thatselectively target and eliminate defined targets, including but notlimited to malignant cells carrying a respective tumor-associatedsurface antigen or virus infected cells carrying a virus-specificsurface antigen or target cells carrying a lineage-specific ortissue-specific surface antigen. Thus, said expression of CARs generatesantigen-specific effector cells for the use in adoptive, target-cellspecific immunotherapy. CARs are composed of a target specificrecognition domain or cell recognition domain (domain (ii), such as ascFv antibody fragment) for recognition of a target (such as atumor-cell surface antigen) fused via a flexible linker region to aneffector domain (comprising a transmembrane region and one or moreintracellular signaling domains like the zeta-chain of the CD3 complexof the T-cell receptor). CAR expression retargets the cytotoxic activityof the effector cells (lymphocytes) to targets (tumor cells) andtriggers their cytolysis by the CAR expressing immune effector cells.Thereby binding of the target specific recognition domain of the CAR toits cognate target on the surface of target cells/viruses transmits asignal into the CAR expressing immune effector cells via theintracellular signaling domain(s) of the CAR which activates theendogenous cytotoxic activity of such imune effector cells.

(i) The Signal Peptide

A “signal peptide” refers to a peptide sequence that directs thetransport and localization of the protein within a cell, e.g. to acertain cell organelle (such as the endoplasmic reticulum) and/or thecell surface.

The signal peptide (i) is a signal peptide of any secreted ortransmembrane human protein of type 1 (extracellular N-terminus), whichallows the transport of the multi-functional protein of the invention tothe cell membrane and cell surface and allows correct localization ofthe multi-functional protein of the invention, in particular theextracellular portion (domains (i) and (ii) and (iii)) on the cellsurface; the transmembrane portion (contributed by/comprised in domain(iv)) inserted into the plasma membrane and the cytoplasmic portion(contributed by/comprised in domain (iv)) in the host cell.

Preferably, the signal peptide is cleaved after passage of theendoplasmic reticulum (ER), i.e. is a cleavable signal peptide.

In an embodiment, the signal peptide (i) comprises or is immunoglobulinheavy chain signal peptide.

(ii) The Target Specific Recognition Domain

The target specific recognition domain (ii) binds an antigen, receptor,peptide ligand or protein ligand of the target.

The target specific recognition domain (ii) preferably comprises

-   -   an antigen binding domain derived from an antibody against an        antigen of the target, or    -   a peptide binding an antigen of the target, or    -   a peptide or protein binding an antibody that binds an antigen        of the target, or    -   a peptide or protein ligand (including but not limited to a        growth factor, a cytokine or a hormone) binding a receptor on        the target, or    -   a domain derived from a receptor (including but not limited to a        growth factor receptor, a cytokine receptor or a hormone        receptor) binding a peptide or protein ligand on the target.

Preferably, the target is a cell or a virus.

The target specific recognition domain serves for the targeting of themulti-functional protein or a respective cell expressing/carrying themulti-functional protein on its surface to a specific target. Binding ofthe target specific recognition domain of the multi-functional protein(CAR) to its cognate target on the surface of target cells/virusesfurthermore transmits a signal into the multi-functional protein(CAR)-expressing immune effector cells via the intracellular signalingdomain(s) of the multi-functional protein which activates the endogenouscytotoxic activity of such imune effector cells.

Preferably, the antigen of the target is

-   -   a tumor-associated surface antigen        -   including but not limited to ErbB2 (HER2/neu),            carcinoembryonic antigen (CEA), epithelial cell adhesion            molecule (EpCAM), epidermal growth factor receptor (EGFR),            EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40,            disialoganglioside GD2, a major histocompatibility complex            (MHC) molecule presenting a tumor-specific peptide epitope,

or

-   -   a lineage-specific or tissue-specific tissue antigen        -   including but not limited to CD3, CD4, CD8, CD24, CD25,            CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86),            endoglin, a major histocompatibility complex (MHC) molecule,

or

-   -   a virus-specific surface antigen,        -   including but not limited to an HIV-specific antigen (such            as HIV gp120), an EBV-specific antigen, a CMV-specific            antigen, a HPV-specific antigen, a HBV-specific antigen, a            HCV-specific antigen, a Lassa Virus-specific antigen, an            Influenza Virus-specific antigen.

In an embodiment, where domain (ii) is derived from an antigen bindingdomain, the antigen binding domain is preferably derived from anantibody or an antibody fragment, such as a single chain Fv (scFv)fragment, a Fab fragment, a diabody, a variable domain of the antibodyheavy chain or antibody light chain.

In an embodiment of the invention, the antigen of the target is thetumor-associated surface antigen ErbB2 and the antigen binding domain ofdomain (ii) is from an ErbB2-specific scFv.

(iii) The Linker Region

The linker region (iii) connects the target specific recognition domain(ii) and the effector domain (iv).

The linker region serves as a flexible spacer between the targetspecific recognition domain (ii) and the effector domain (iv). Itensures the necessary accessibility and flexibility of the targetspecific recognition domain (ii). The linker region is understood to beessential for the functionality of the multi-functional proteins of theinvention.

Current CAR constructs contain a linker region derived from thealpha-chain of the murine or human CD8 molecule which provides aflexible connection between cell-targeting and signaling/effectordomains (Uherek et al., 2002; Müller et al., 2008). However, unpairedcysteine(s) present in the linker region of the CD8 alpha-chain canresult in unwanted covalent intra- or intermolecular bonds of CARmolecules which negatively affects surface expression of CAR as well asCAR functionality.

Object matter of the invention is the generation of optimized CARconstructs which do not form such non-productive covalent linkages viathe unpaired cysteine residue of the human CD8 alpha-chain andfacilitate efficient surface expression and high functionality inlymphocytes.

This is achieved, according to the invention, by employing a specificfragment of the hinge region derived from the human CD8 alpha-chainranging from amino acid positions 117 to 178 (numbering according to thesequence of human T-cell surface glycoprotein CD8 alpha chain;Swiss-Prot accession number P01732), and by modifying the amino acidsequence of the hinge region derived from the human CD8 alpha-chain, inparticular by replacing/converting the unpaired cysteine(s) to (a)serine residues or by deleting the unpaired cysteine(s). The resultingoptimized CAR construct is expressed at higher levels at the cellsurface and mediates more potent antigen-specific killing. In comparisonto cells carrying a current CAR, cells carrying the optimized CARconstruct contain a lower level of unpaired endogenous effector domain(such as CD3 zeta-chain) but higher levels of functional receptorcomplexes and productive dimers between CAR and endogenous effectordomain (such as CD3 zeta-chain).

In particular, the linker region (iii) comprises a modified hinge regionof the human CD8 alpha-chain.

The sequence of human T-cell surface glycoprotein CD8 alpha chain(Swiss-Prot accession number P01732 (CD8A_HUMAN)) [SEQ ID NO. 13]

        10         20         30         40MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE        50         60         70         80LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA        90        100        110        120AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN       130        140        150        160SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR       170        180        190        200PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL        210        220        230 VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYVwherein the flexible hinge region are amino acid residues 117 to 178[SEQ ID NO. 1]:

ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA C RPAAGGAVHTRGLD

The modification of the human CD8 alpha-chain hinge region according tothe invention is the replacement of the cysteine residue(s) with (a)serine residue(s) or the deletion of the cysteine residue(s).

According to the invention, the linker region (iii) consists of theamino acid sequence of SEQ ID NO. 2:

ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA S RPAAGGAVHTRGLD

or an amino acid sequence that has at least 95% sequence identity or 99%sequence identity to the amino acid sequence of SEQ ID NO. 2, under theproviso that amino acid residue no. 48 of SEQ ID NO. 2 is not a cysteineand is a serine and under the proviso that the amino acid sequence doesnot contain any cysteine residue(s),

or an amino acid sequence that differs in one, two or three amino acidresidues from the amino acid sequence of SEQ ID NO. 2, under the provisothat amino acid residue no. 48 of SEQ ID NO. 2 is not a cysteine and isa serine and under the proviso that the amino acid sequence does notcontain any cysteine residue(s), wherein “differ” refers toreplacement/substitution, addition or deletion, such as conservativesubstitution(s) of amino acid residues.

Thus, the linker region (iii) does not contain any cysteine residue(s).

Thus, the linker region (iii) is selected from any of the following:

-   -   the amino acid sequence of SEQ ID NO. 2,    -   an amino acid sequence with at least 95% sequence identity to        SEQ ID NO. 2 under the proviso that amino acid residue 48 is not        a cysteine and is a serine,

and

-   -   an amino acid sequence that differs in one, two or three amino        acid residues from the amino acid sequence of SEQ ID NO. 2 under        the proviso that amino acid residue 48 is not a cysteine and is        a serine.

As discussed above, prior art describes chimeric antigen receptors thatcontain as linker regions different fragments of the hinge regionderived from the human or murine CD8 alpha-chain. However, the specificmodified hinge region of the invention that is used as the linker region(iii) in the multi-functional proteins according to the invention hasnot been used or disclosed in the art and has been found by theinventors to be particularly advantageous for the expression of themulti-functional proteins/CARs according to the invention and theirtransport to the surface of the effector cells (as has been demonstratedin this specification e.g. in FIGS. 3A-3B and 4). Furthermore, thespecific modified hinge region of the invention results in improvedfunctionality of the multi-functional proteins/CARs according to theinvention (as has been demonstrated in this specification e.g. in FIGS.4 and 5C). This improved expression and functionality of CARs accordingto the invention is due to the selection and specific modification ofamino acid residues 117 to 178 from the human CD8 alpha-chain as thelinker region (iii) in the multi-functional proteins. The specificmodified hinge region of the invention that is used as the linker region(iii) in the multi-functional proteins according to the inventionprevents the occurence of unpaired cysteines by not including thecysteines naturally present at amino acid positions 115 and 181 of thehuman CD8 alpha-chain, and replacement of the cysteine residue naturallypresent at amino acid position 164 of the human CD8 alpha-chain with achemically similar serine residue. Furthermore, the length of 62 aminoacid residues of the specific modified hinge region of the inventionthat is used as the linker region (iii) in the multi-functional proteinsaccording to the invention ensures optimal spatial distance of theN-terminally attached target specific recognition domain (ii) from theC-terminally attached transmembrane and intracellular effector domain(iv), providing high flexibility and efficiency of target cellrecognition. In contrast, prior art describes CARs employing as linkerregions unmodified fragments of the hinge region from the human CD8alpha-chain (see, for example, Fitzer-Attas et al. 1998, WO 2008/045437)or murine CD8 alpha-chain (see, for example, WO 95/30014) that containnaturally occurring cysteines of the CD8 alpha-chain, which cannegatively affect expression and functionality of these CARs through theformation of undesired intra- or intermolecular disulfide bonds.Furthermore, prior art describes CARs employing as linker regionsmodified fragments of the hinge region from the human CD8 alpha-chainthat encompass significantly shorter amino acid sequences (such as onlyabout 30 to about 40 amino acid residues, see, for example, US2007/0031438), which reduces spatial distance of the target specificrecognition domain from the effector domain and can negatively affectflexibility and efficiency of target cell recognition.

(iv) The Effector Domain

The effector domain (iv) comprises a transmembrane region and one ormore intracellular signaling domains.

The effector domain serves the coupling of the target/antigenrecognition to the intracellular signaling machinery.

Binding of the target specific recognition domain (ii) of themulti-functional protein (CAR) to its cognate target on the surface oftarget cells/viruses furthermore transmits a signal into themulti-functional protein (CAR)-expressing immune effector cells via theintracellular signalling domain(s) of the multi-functional protein(which are part of the effector domain) which activates the endogenouscytotoxic activity of such immune effector cells.

Preferably, the effector domain (iv) comprises or consists of (is)

-   -   (a) the zeta-chain of the human CD3 complex of the T-cell        receptor or fragment(s) thereof; or a functional equivalent        thereof,    -   (b) a fusion of a fragment of the human costimulatory CD28        receptor fused to a fragment of the zeta-chain of the human CD3        complex of the T-cell receptor, preferably a fusion of the        transmembrane and intracellular domain of human CD28 with the        intracellular domain of human CD3 zeta chain; or a functional        equivalent thereof.

The term “functional equivalent” defines a protein or nucleotidesequence, having a different amino acid or base sequence, compared tothe sequences disclosed herein, but exhibiting the same function invitro and in vivo. An example of a functional equivalent is a modifiedor synthetic gene, encoding the expression of a protein identical orhighly homologous to that encoded by the wildtype gene or a sequencedisclosed herein.

-   (a) The sequence of human T-Cell surface glycoprotein CD3 zeta chain    (Swiss-Prot accession number P20963 (CD3Z_HUMAN); Isoform 3) [SEQ ID    NO. 3]

        10         20         30         40MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF        50         60         70         80IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR        90        100        110        120EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE       130        140        150        160AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR

The effector domain (iv) comprises or consists of (is) an amino acidsequence with SEQ ID NO. 3 or fragment(s) thereof (preferably thetransmembrane and intracellular domain of human CD3 zeta-chain, morepreferably amino acid residues 29 to 163 of amino acid sequence with SEQID NO. 3) or a functional equivalent thereof, wherein a “functionalequivalent” has less sequence identity (such as at least 80% sequenceidentity, preferably at least 90% sequence identity, more preferably atleast 95% sequence identity or 99% sequence identity) but is afunctional zeta-chain of the CD3 complex of the T-cell receptor.

According to the invention, the zeta chain is of human origin. Withinthe TCR the CD3 zeta chain exists as a disulfide homodimer. A“functional CD3 zeta chain” or “a functional zeta-chain of the CD3complex of the T-cell receptor” is a protein which upon expression in Tcell hybridomas deficient in endogenous zeta expression is capable ofrestoring in said hybridomas a functionally active TCR.

-   (b) the Fusion of a Fragment of the Costimulatory CD28 Receptor    Fused to a Fragment of the Zeta-Chain of the CD3 Complex of the    T-Cell Receptor Contains:

(b1) the transmembrane domain of human CD28;

(b2) the intracellular domain of human CD28;

(b3) the intracellular domain of human CD3 zeta chain;

The sequence of human T-cell-specific surface glycoprotein CD28(Swiss-Prot accession number P10747 (CD28_HUMAN)) [SEQ ID NO. 4]

        10         20         30         40MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC        50         60         70         80KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS        90        100        110        120KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP       130        140        150        160PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG       170        180        190        200GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG        210        220PTRKHYQPYA PPRDFAAYRSwherein (b1) are preferably amino acid residues 151-180 of SEQ ID NO. 4,(b2) are amino acid residues 181-220 of SEQ ID NO. 4 and (b3) are aminoacid residues 52-163 of SEQ ID NO. 3 (=SEQ ID NO. 5):

KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS RVKFSRSADAPAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPRRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR

The effector domain (iv) comprises or consists of (is) an amino acidsequence with the amino acid sequence of SEQ ID NO. 5 or a functionalequivalent thereof, wherein a “functional equivalent” has less sequenceidentity (such as at least 80% sequence identity, preferably at least90% sequence identity, more preferably at least 95% sequence identity or99% sequence identity) but is a functional fusion of the costimulatoryCD28 receptor fused to a fragment of the zeta-chain of the CD3 complexof the T-cell receptor.

Preferably, the multi-functional protein according to the inventioncomprises or consists of the amino acid sequence of a (cleavable) signalpeptide (i), an scFv (ii), the modified hinge region (iii) (as definedherein, preferably of SEQ ID NO. 2) and the CD3 zeta chain orfragment(s) thereof or a fusion of fragment(s) of CD28 with fragment(s)of CD3 zeta-chain (iv) (wherein the signal peptide is at the N-terminusand the zeta chain/fusion is at the C-terminus).

In a preferred embodiment, the protein comprises or consists of theamino acid sequence of SEQ ID NO. 6; or an amino acid sequence that hasat least 95% sequence identity or 99% sequence identity to the aminoacid sequence of SEQ ID NO. 6 (under the proviso that amino acid residueno. 308 (i.e. amino acid residue no. 48 of the modified hinge region(SEQ ID NO. 2)) is not a cysteine and is a serine and under the provisothat the amino acid sequence of the modified hinge region (i.e. aminoacid residues no. 261 to 322) does not contain any cysteine residue(s).

The amino acid sequence of SEQ ID NO. 6 refers to the amino acidsequence of the multi-functional protein with the domains:

-   (i) [signal peptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified    hinge]-(iv)[transmembrane and intracellular domain of the human CD3    zeta chain]

MDWIWRILFLVGAATGAHSQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYF CQQHFRTPFTFGSGTKLEIKALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDPKLCYLLDGILFIY GVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In a preferred embodiment, the protein comprises or consists of theamino acid sequence of SEQ ID NO. 7; or an amino acid sequence that hasat least 95% sequence identity or 99% sequence identity to the aminoacid sequence of SEQ ID NO. 7 (under the proviso that amino acid residueno. 308 (i.e. amino acid residue no. 48 of the modified hinge region(SEQ ID NO. 2)) is not a cysteine and is a serine and under the provisothat the amino acid sequence of the modified hinge region (i.e. aminoacid residues no. 261 to 322) does not contain any cysteine residue(s).

The amino acid sequence of SEQ ID NO. 7 refers to the amino acidsequence of the multi-functional protein with the domains:

-   (i)[signal peptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified    hinge]-(iv)[fusion of the transmembrane and intracellular domain of    human CD28 with the intracellular domain of human CD3 zeta chain].

MDWIWRILFLVGAATGAHSQVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDSATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYF CQQHFRTPFTFGSGTKLEIKALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Generally, a person skilled in the art is aware of the fact that someamino acid exchanges in the amino acid sequence of a protein or peptidedo not have any influence on the (secondary or tertiary) structure,function and activity of the protein or peptide (at all). Amino acidsequences with such “neutral” amino acid exchanges as compared to theamino acid sequences disclosed herein fall within the scope of thepresent invention.

Nucleic Acids, Expression Constructs and Host Cells

As described above, the present invention provides nucleic acids/nucleicacid molecules/isolated nucleic acid molecules encoding the proteins ofthe invention.

The nucleic acids according to this invention comprise DNA (such asdsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinationsthereof or derivatives (such as PNA) thereof.

Preferably, a nucleic acid of the invention comprises

-   -   the nucleic acid encoding for the amino acid sequence of SEQ ID        NO: 2;    -   or    -   the nucleic acid sequence of SEQ ID NO: 8 (=nucleotide sequence        encoding for the modified hinge region)    -   or their complementary sequences;    -   or sequences that have at least 95% sequence identity or 99%        sequence identity to the above sequences (provided that amino        acid residue no. 48 of SEQ ID NO: 2 is not a cysteine and is a        serine and provided that the modified hinge region does not        contain any cysteine residues).

Preferably, a nucleic acid of the invention furthermore comprises

-   -   the nucleic acid encoding for the amino acid sequence of SEQ ID        NOs: 3 or 5 or for amino acid residues 29-163 of SEQ ID NO: 3;    -   or    -   the nucleic acid sequence of SEQ ID NOs: 9 or 10 (=nucleotide        sequence encoding for the transmembrane and intracellular domain        of human CD3 zeta chain or the fusion of the transmembrane and        intracellular domain of human CD28 with the intracellular domain        of human CD3 zeta chain),    -   or their complementary sequences;    -   or sequences that have at least 95% sequence identity or 99%        sequence identity to the above sequences,

preferably fused to the nucleic acid encoding for the amino acidsequence of SEQ ID NO: 2 or the nucleic acid sequence of SEQ ID NO: 8

-   -   or to their complementary sequences;    -   or to sequences that have at least 95% sequence identity or 99%        sequence identity to the above sequences SEQ ID NO: 2 or SEQ ID        NO: 8 (provided that amino acid residue no. 48 of SEQ ID NO: 2        is not a cysteine and is a serine and provided that the modified        hinge region does not contain any cysteine residues).

Preferably, a nucleic acid of the invention comprises or consists of

-   -   the nucleic acid encoding for the amino acid sequence of SEQ ID        NOs: 6 or 7;    -   or    -   the nucleic acid sequence of SEQ ID NOs: 11 or 12 (=nucleotide        sequence encoding for the multi-functional protein with the        domains (i)[signal peptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified        hinge]-(iv)[transmembrane and intracellular domain of the human        CD3 zeta chain] or the multi-functional protein with the domains        (i)[signal peptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified        hinge]-(iv)[fusion of the transmembrane and intracellular domain        of human CD28 with the intracellular domain of human CD3 zeta        chain]);    -   or their complementary sequences;    -   or sequences that have at least 95% sequence identity or 99%        sequence identity to the above sequences (provided that amino        acid residue no. 48 of SEQ ID NO: 2 is not a cysteine and is a        serine and under the proviso that the amino acid sequence of the        modified hinge region (i.e. amino acid residues no. 261 to 322)        does not contain any cysteine residue(s)).

Preferably, the nucleic acid sequences of the present invention arecodon-optimized for expression in mammalian cells, preferably forexpression in human cells. Codon-optimization refers to the exchange ina sequence of interest of codons that are generally rare in highlyexpressed genes of a given species by codons that are generally frequentin highly expressed genes of such species, such codons encoding the sameamino acids as the codons that are being exchanged.

Within the scope of this invention are also the nucleotide sequencesobtained due to the degeneration of the genetic code of the abovenucleotide sequences.

As described above, the present invention provides expression constructsfor expressing the protein of the invention in a cell.

Preferably, the expression constructs further comprise promoter andterminator sequences.

An “expression or gene construct” (wherein both terms are usedinterchangeably throughout this specification) refers to a nucleic acidconstruct, usually an expression vector or plasmid, that is used tointroduce a specific gene sequence into a target cell. Once theexpression or gene construct is inside the cell, the protein that isencoded by the gene is produced by the cellular transcription andtranslation machinery. The expression or gene construct is designed tocontain respective regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the construct, including promoter and terminator sequences. The goalof a well-designed expression or gene construct is the production oflarge amounts of stable mRNA, and therefore proteins.

The skilled artisan can select further suitable components of expressionor gene constructs.

The nucleic acids and/or in particular expression constructs of theinvention are capable of directing the synthesis/expression of themulti-functional protein of the invention in a suitable host cell.

The nucleic acids and/or expression constructs of the invention aredsDNA, ssDNA, RNA or mRNA or combinations thereof.

As described above, the present invention provides host cells whichexpress a protein of the invention or which comprise a nucleic acid oran expression construct of the invention.

Preferably, the host cell is selected from effector cells of the immunesystem, such as lymphocytes including but not limited to cytotoxiclymphocytes, T cells, cytotoxic T cells, T helper cells, Th17 T cells,natural killer (NK) cells, natural killer T (NKT) cells, mast cells,dendritic cells, killer dendritic cells, B cells.

“Effector cells” of the immune system or “immune effector cells” refersto cells of hematopoietic origin including but not limited to the celltypes mentioned above that are functionally involved in the initiationand/or execution of innate and/or adaptive immune responses.

Uses of the Proteins, Nucleic Acids, Expression Constructs and HostCells

As described above, the invention provides the use of themulti-functional protein, nucleic acid, or expression construct forgenerating antigen-specific effector cells.

“Antigen-specific effector cells” or “target-specific effector cells”refer to effector cells of the immune system or immune effector cellsgenetically modified to express the multi-functional protein of theinvention by transfer of an expression construct or nucleic acidencoding said multi-functional protein. Such antigen-specific ortarget-specific effector cells are versatile means, in particular in thetreatment of diseases (as described below for ACT and cancer treatment).

As described above, the invention provides the multi-functional protein,nucleic acid, expression construct or host cell for use as a medicament.

As described above, the invention provides the multi-functional protein,nucleic acid, expression construct or host cell for use in the treatmentof cancer.

As described above, the invention provides the multi-functional protein,nucleic acid, expression construct or host cell for use in adoptive,target-cell specific immunotherapy.

“Adoptive, target-cell specific immunotherapy” refers to a form oftherapy in which immune cells are transferred to tumor-bearing hosts.The immune cells have antitumor reactivity and can mediate direct orindirect antitumor effects.

“Adoptive, target-cell specific immunotherapy” or “adoptive cell therapy(ACT)” is a treatment that uses immune effector cells, such aslymphocytes with anti-tumour activity, expanded in vitro and infusedinto the patient with cancer. ACT using autologous tumour-infiltratinglymphocytes has emerged as the most effective treatment for patientswith metastatic melanoma and can mediate objective cancer regression inapproximately 50% of patients. The use of donor lymphocytes for ACT isan effective treatment for immunosuppressed patients who developpost-transplant lymphomas (reviewed in Rosenberg et al., 2008). However,the ability to genetically engineer human lymphocytes and use them tomediate cancer regression in patients, which has recently beendemonstrated (see Morgan et al, 2006), has opened possibilities for theextension of ACT immunotherapy to patients with a wide variety of cancertypes and is a promising new approach to cancer treatment. Thus,genetically engineering of lymphocytes with chimeric antigen receptors(CAR), such as provided by this invention, is very suitable for ACT andopens more possibilities in the treatment of cancer. Especially, sincestudies have clearly demonstrated that the administration of highly avidanti-tumour T cells directed against a suitable target can mediate theregression of large, vascularized, metastatic cancers in humans andprovide guiding principles as well as encouragement for the furtherdevelopment of immunotherapy for the treatment of patients with cancer.

Methods of Treatment

Furthermore, the invention provides methods for generatingantigen-specific effector cells.

The method for generating antigen-specific effector cells according tothe present invention comprises

-   -   (a) providing a multi-functional protein, nucleic acid, or        expression construct according to the invention;    -   (b) providing a host cell or cell line, which is selected from        effector cells of the immune system, such as lymphocytes        including but not limited to cytotoxic lymphocytes, T cells,        cytotoxic T cells, T helper cells, Th17 T cells, natural killer        (NK) cells, natural killer T (NKT) cells, mast cells, dendritic        cells, killer dendritic cells, B cells;    -   (c) transferring the multi-functional protein, nucleic acid, or        expression construct provided in step (a) into the host cell or        cell line provided in step (b);    -   (d) optional, selection of the transgenic (gene-modified) cells.

The present invention also provides methods for the treatment ofdiseases, in particular cancer, and methods of immunotherapy, preferablyincluding adoptive, target-cell specific immunotherapy.

The method for the treatment of diseases, in particular cancer,according to the present invention comprises

administering to a subject in a therapeutically effective amount

-   -   (a) a multi-functional protein, a nucleic acid, an expression        construct or a host cell (in particular an antigen-specific        effector cell) as obtained and defined herein, and    -   (b) optionally, respective excipient(s).

The method of immunotherapy, preferably including or utilizing adoptive,target-cell specific immunotherapy, according to the present inventioncomprises

administering to a subject in a therapeutically effective amount

-   -   (a) a multi-functional protein, a nucleic acid, an expression        construct or a host cell (in particular an antigen-specific        effector cell) as obtained and defined herein, and    -   (b) optionally, respective excipient(s).

A “therapeutically effective amount” of multi-functional protein, anucleic acid, an expression construct or a host cell (in particular anantigen-specific effector cell) of this invention refers to the amountthat is sufficient to treat the respective disease or achieve therespective outcome of the adoptive, target-cell specific immunotherapy.

SEQUENCES

SEQ ID NO:1 shows the amino acid sequence of the hinge region of humanT-cell surface glycoprotein CD8 alpha chain (amino acid residues 117-178of SEQ ID NO. 13).

SEQ ID NO:2 shows the amino acid sequence of the modified hinge regionderived from the human CD8 alpha-chain hinge region.

SEQ ID NO:3 shows the amino acid sequence of human T-cell surfaceglycoprotein CD3 zeta chain (Swiss-Prot accession number P20963(CD3Z_HUMAN); Isoform 3.

SEQ ID NO:4 shows the amino acid sequence of human T-cell-specificsurface glycoprotein CD28 (Swiss-Prot accession number P10747(CD28_HUMAN).

SEQ ID NO:5 shows the amino acid sequence of the fusion of thetransmembrane domain and the intracellular domain of human CD28 (aminoacid residues 151-220 of SEQ ID NO. 4) and the intracellular domain ofhuman CD3 zeta chain (amino acid residues 52-163 of SEQ ID NO:3).

SEQ ID NO:6 shows the amino acid sequence of the multi-functionalprotein with the domains (i)[signal peptide]-(ii)[anti-ErbB2scFv]-(iii)[modified hinge]-(iv)[transmembrane and intracellular domainof the human CD3 zeta chain].

SEQ ID NO:7 shows the amino acid sequence of the multi-functionalprotein with the domains (i)[signal peptide]-(ii)[anti-ErbB2scFv]-(iii)[modified hinge]-(iv)[fusion of the transmembrane andintracellular domain of human CD28 with the intracellular domain ofhuman CD3 zeta chain].

SEQ ID NO:8 shows the nucleotide sequence encoding for the modifiedhinge region in a codon-optimized form.

SEQ ID NO:9 shows the nucleotide sequence encoding for transmembranedomain and the intracellular domain of human CD3 zeta chain in acodon-optimized form.

SEQ ID NO:10 shows the nucleotide sequence encoding for the fusion ofthe transmembrane and intracellular domain of human CD28 with theintracellular domain of human CD3 zeta chain in a codon-optimized form.

SEQ ID NO:11 shows the nucleotide sequence encoding for themulti-functional protein with the domains (i)[signalpeptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified hinge]-(iv)[transmembraneand intracellular domain of the human CD3 zeta chain] in acodon-optimized form.

SEQ ID NO:12 shows the nucleotide sequence encoding for themulti-functional protein with the domains (i)[signalpeptide]-(ii)[anti-ErbB2 scFv]-(iii)[modified hinge]-(iv)[fusion of thetransmembrane and intracellular domain of human CD28 with theintracellular domain of human CD3 zeta chain] in a codon-optimized form.

SEQ ID NO:13 shows the amino acid sequence of human T-cell surfaceglycoprotein CD8 alpha chain (Swiss-Prot accession number P01732(CD8A_HUMAN)).

The following examples and drawings illustrate the present inventionwithout, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B Modified hinge region derived from CD8 alpha-chain.

(1A) Amino acid sequences of original (SEQ ID NO:1) and (1B) modifiedhinge regions (SEQ ID NO:2) derived from human CD8 alpha-chain areshown. The unpaired cysteine and the modified residue are underlined.

FIG. 2 Schematic representation of the expression construct.

The sequence encoding the ErbB2-specific CAR is expressed under thecontrol of a Spleen Focus Forming Virus promoter (SFFV) and followed byan internal ribosome entry site (IRES) and cDNA encoding enhanced greenfluorescent protein (EGFP). The CAR is composed of an immunoglobulinheavy chain signal peptide (SP), an ErbB2-specific single-chain Fvantibody fragment (scFv), unmodified or modified CD8 alpha-chain hingeregion as a flexible linker (CD8 alpha), and the transmembrane domainand the intracellular domain of CD3 zeta-chain as a signaling domain(zeta).

FIGS. 3A-3B Analysis of CAR surface expression in transduced NK cells.

NK cells were transduced with lentiviral vectors encoding ErbB2-specificCAR containing either unmodified (upper panel, dark gray (3A)) ormodified CD8 alpha-chain hinge region (lower panel, dark gray (3B)).Gene-modified cells were selected by FACS-based sorting. Expression ofCAR on the surface of NK cells was investigated by FACS analysis usingErbB2-Fc fusion protein. NK cells transduced with empty vector served ascontrol (light gray).

FIG. 4 Immunoblot analysis of CAR expression.

Lysates of transduced NK cells expressing ErbB2-specific CAR eithercontaining the modified (lane 2) or unmodified CD8 alpha-chain hingeregion (lane 3) were subjected to SDS-PAGE under non-reducing conditionsand immunoblot analysis with anti-CD3 zeta-chain antibody as indicated.Lysate of untransduced NK cells served as control (lane 1). Monomers andhomodimers of endogenous CD3 zeta-chain, CAR-CD3 zeta-chainheterodimers, and CAR homodimers are indicated.

FIGS. 5A-5C Cytotoxic activity of CAR-expressing NK cells.

NK cells expressing ErbB2-specific CAR either containing the modified orunmodified CD8 alpha-chain hinge region were co-cultured at differenteffector to target (E:T) ratios with NK-sensitive but ErbB2-negativeK562 erythroleukemic control cells (5A), ErbB2-negative MDA-MB468 breastcarcinoma cells (5B), or ErbB2-positive MDA-MB453 breast carcinoma cells(5C). As shown in (5C), NK cells expressing the ErbB2-specific CAR withthe modified CD8 alpha-chain hinge region showed markedly enhancedErbB2-specific cell killing (open bars) when compared to NK cellsexpressing the ErbB2-specific CAR with unmodified CD8 alpha-chain hingeregion (filled bars).

FIGS. 6A-6D NK cells expressing a CAR that contains CD28 and CD3 zetachain domains.

(6A) The sequence encoding the ErbB2-specific CAR is expressed under thecontrol of a Spleen Focus Forming Virus promoter (SFFV) and followed byan internal ribosome entry site (IRES) and cDNA encoding enhanced greenfluorescent protein (EGFP). The CAR is composed of an immunoglobulinheavy chain signal peptide (SP), an ErbB2-specific single-chain Fvantibody fragment (scFv), the modified CD8 alpha-chain hinge region as aflexible linker (CD8 alpha), and CD28 and CD3 zeta-chain (zeta) assignaling domains. (6B) NK cells were transduced with the lentiviralvector shown in (6A). Gene-modified cells were selected by FACS-basedsorting. Expression of CAR on the surface of NK cells was investigatedby FACS analysis using ErbB2-Fc fusion protein (dark gray).Non-transduced NK cells served as control (light gray). NK cellsexpressing ErbB2-specific CAR containing the modified CD8 alpha-chainhinge region and CD28 and CD3 zeta-chain as signaling domains wereco-cultured at different effector to target (E:T) ratios withErbB2-negative MDA-MB468 breast carcinoma cells (6C), or ErbB2-positiveMDA-MB453 breast carcinoma cells (6D). As shown in (6D), NK cellsexpressing the ErbB2-specific CAR with the modified CD8 alpha-chainhinge region and CD28 and CD3 zeta-chain as signaling domains showedErbB2-specific cell killing (open bars) when compared to non-transducedNK cells included as control (filled bars).

EXAMPLES Example 1

Construction of CAR. A cDNA fragment encoding the hinge region derivedfrom the human CD8 alpha-chain was mutated by site-directed mutagenesisto replace the codon encoding the unpaired cysteine of the hinge regionto a codon encoding a serine residue (FIGS. 1A-1B). Sequences encodingan immunoglobulin heavy chain signal peptide, a scFv antibody fragmentspecific for the tumor-associated surface antigen ErbB2, the modifiedhinge region derived from human CD8 alpha-chain, and transmembrane andintracellular domains of human CD3 zeta-chain were assembled into asingle open reading frame resulting in an ErbB2-specific CAR encodingcDNA. The CAR encoding sequence was inserted into the lentiviraltransfer vector SIEW for expression in lymphocytes under the control ofthe Spleen Focus Forming Virus promoter (FIG. 2). For comparison alentiviral transfer vector was produced encoding a similar CARcontaining the unmodified hinge region derived from the human CD8alpha-chain.

Transduction of NK cells. VSV-G pseudotyped lentiviral vector particleswere produced by transient triple transfection of 293T cells with thetransfer vector together with the packaging constructs pMD-VSVG and8.91. Lentiviral vector was used for transduction of NK cells, andtransduced NK cells were enriched by two rounds of FACS sorting based onexpression of enhanced green fluorescent protein (EGFP) as a marker geneencoded by the SIEW vector.

Surface expression of CAR. Expression of CAR on the surface oftransduced and FACS-sorted NK cells was investigated by FACS analysiswith an ErbB2-Fc fusion protein (R&D Systems) followed by APC-conjugatedanti-human Fc F(ab)₂ fragment. NK cells transduced with CAR containingthe modified CD8 alpha-chain hinge region displayed a higher overallsurface expression of CAR when compared to NK cells expressing a similarCAR containing the unmodified CD8 alpha-chain hinge region (FIGS.3A-3B).

Immunoblot analysis of CAR expression. CAR expression andmultimerization in transduced and FACS-sorted NK cells was investigatedby immunoblot analysis. Proteins in cell lysates of transduced cellswere separated by SDS-PAGE under non-reducing conditions. Subsequentimmunoblot analysis with anti-CD3 zeta-chain antibody demonstrated amarked reduction in the level of unpaired endogenous zeta-chain andhigher levels of CAR-zeta-chain heterodimers and CAR homodimers insamples from NK cells expressing CAR with the modified CD8 alpha-chainhinge region when compared to NK cells expressing a similar CARcontaining the unmodified CD8 alpha-chain hinge region (FIG. 4).

Cytotoxic activity of CAR-expressing NK cells. The cytotoxic activity ofCAR-expressing NK cells was measured in FACS-based cytotoxicity assays.NK cells expressing ErbB2-specific CAR either containing the modified orunmodified CD8 alpha-chain hinge region displayed similar cytotoxicactivity towards NK-sensitive but ErbB2-negative K562 erythroleukemiccontrol cells, but were both unable to lyse NK-resistant andErbB2-negative MDA-MB468 breast carcinoma cells. When cytotoxic activitytowards ErbB2-positive MDA-MB453 breast carcinoma cells was tested, NKcells expressing the ErbB2-specific CAR with the modified CD8alpha-chain hinge region showed markedly enhanced ErbB2-specific cellkilling when compared to NK cells expressing the ErbB2-specific CAR withunmodified CD8 alpha-chain hinge region (FIGS. 5A-5C). These resultsdemonstrate that the modified CAR posseses enhanced functionality.

Example 2

Construction of CAR containing CD28 and CD3 zeta-chain signaling domainsSequences encoding an immunoglobulin heavy chain signal peptide, a scFvantibody fragment specific for the tumor-associated surface antigenErbB2, the modified hinge region derived from human CD8 alpha-chain asdescribed in Example 1, transmembrane and intracellular domains of humanCD28, and the intracellular domain of human CD3 zeta-chain wereassembled into a single open reading frame resulting in anErbB2-specific CAR encoding cDNA containing CD28 and CD3 zeta-chainsignaling domains. The CAR encoding sequence was inserted into thelentiviral transfer vector SIEW for expression in lymphocytes under thecontrol of the Spleen Focus Forming Virus promoter (FIG. 6A).

Transduction of NK cells. VSV-G pseudotyped lentiviral vector particleswere produced by transient triple transfection of 293T cells with thetransfer vector together with the packaging constructs pMD-VSVG and8.91. Lentiviral vector was used for transduction of NK cells, andtransduced NK cells were enriched by two rounds of FACS sorting based onexpression of enhanced green fluorescent protein (EGFP) as a marker geneencoded by the SIEW vector.

Surface expression of CAR containing CD28 and CD3 zeta-chain signalingdomains. Expression of CAR containing CD28 and CD3 zeta-chain signalingdomains on the surface of transduced and FACS-sorted NK cells wasinvestigated by FACS analysis with an ErbB2-Fc fusion protein (R&DSystems) followed by APC-conjugated anti-human Fc F(ab)₂ fragment. NKcells transduced with CAR containing the modified CD8 alpha-chain hingeregion and CD28 and CD3 zeta-chain signaling domains displayed highsurface expression of CAR (FIG. 6B).

Cytotoxic activity of NK cells expressing a CAR that contains CD28 andCD3 zeta-chain signaling domains. The cytotoxic activity of NK cellsexpressing a CAR that contains the modified CD8 alpha-chain hinge regionand CD28 and CD3 zeta-chain signaling domains was measured in FACS-basedcytotoxicity assays. NK cells expressing this ErbB2-specific CAR andcontrol NK cells not expressing a CAR were both unable to lyseNK-resistant and ErbB2-negative MDA-MB468 breast carcinoma cells (FIG.6C). When cytotoxic activity towards ErbB2-positive MDA-MB453 breastcarcinoma cells was tested, NK cells expressing the ErbB2-specific CARwith the modified CD8 alpha-chain hinge region and CD28 and CD3zeta-chain signaling domains showed high ErbB2-specific cell killingwhereas control NK cells not expressing a CAR were unable to lyse thetarget cells to a significant degree (FIG. 6D). These resultsdemonstrate that the functionality of the modified CD8 alpha-chain hingeregion is retained as part of a CAR that contains CD28 and CD3zeta-chain signaling domains.

Materials and Methods (for Examples 1 and 2)

Cells and culture conditions. Human NK cells were maintained in X-VIVO10medium supplemented with 5% human plasma and 100 IU/mL IL-2.

Production of VSV-G pseudotyped vectors in 293T cells. Vector particleswere generated by transient transfection of 4×10⁶ HEK-293T cells with athree plasmid system consisting of the packaging plasmid coding for theVSV-G envelope protein (pMD-VSVG), the glycoprotein expression plasmidencoding gag and pol (8.91), and the transfer plasmid carrying the geneof interest. Cells were transfected by calcium phosphate transfectionusing a total of 20 μg plasmid DNA consisting of 6.5 μg gag pol, 3.5 μgVSV-G, and 10 μg of transfer plasmids. DNA-calciumphosphate-precipitates were added dropwise to cell monolayers, and 10 mMchloroquine were added. Cell culture supernatants containing pseudotypedlentiviral vector particles were harvested 48 h later. Supernatants weresterile filtered (0.45 μm filter) and directly used for transduction ofNK cells.

Lentiviral transduction. For transduction, 5×10⁵ NK cells were seededinto a single well of a 6 well plate. Vector particles were added to thecells in the presence of 8 μg/mL polybrene and centrifuged for 60 min at1800 rpm at 32° C. 48 h after transduction the cells were analyzed byFACS for EGFP and CAR expression.

Flow cytometric analysis. For analysis of CAR expression, transduced NKcells were incubated with 1 μg ErbB2-Fc fusion protein (R&D Systems) for1 h at 4° C. Then cells were washed and stained with a secondaryAPC-coupled anti-human Fc F(ab)₂ antibody fragment for 20 min at 4° C.Samples were washed in FACS buffer (DPBS, 3% FCS) and resuspended in 250μl for FACS analysis using a FACSCanto flow cytometer (BD Biosciences).Non-transduced NK cells or NK cells transduced with empty SIEWlentiviral vector served as control.

Immunoblot analysis. 5×10⁶ NK cells were harvested and pelleted. Afterwashing twice with DPBS, 500 μL lysis buffer (20 mM Tris, pH 7.3, 137 mMNaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, protease inhibitors)were added to the cell pellet and incubated for 20 min on ice. Celldebris was removed by centrifugation at 14,000 rpm for 10 min at 4° C.Lammli buffer without addition of reducing reagents was added to thecleared supernatants, and the samples were subjected to SDS-PAGE andimmunoblot analysis with anti-CD3 zeta-chain antibody following standardprocedures.

FACS-based cytotoxicity assays. To investigate cytotoxic activity ofparental and genetically modified NK cells (effector cells, E) towardsdifferent tumor cell lines (target cells, T), a FACS-based cytotoxicityassay was used. Target cells were labeled with calcein violet AM(Molecular Probes, Invitrogen). Cells were harvested, counted and washedin calcein wash buffer (RPMI1640). The cell number was adjusted to 4×10⁶cells/mL, and 1.5 calcein violet AM dissolved in 42 μL DMSO were addedto the cells. Staining of cells was performed for 30 min on ice. Thencells were washed three times with calcein wash buffer, and the cellnumber was adjusted to 5×10⁵ cells/mL. To test cytotoxic activity ofgenetically modified NK cells, effector and labeled target cells wereco-cultured at various effector to target (E/T) ratios. First, effectorcells were pelleted, counted and the cell number was adjusted to 5×10⁶cells/mL. Appropriate dilutions were prepared. For co-cultureexperiments target cells were resuspended in X-VIVO medium containing 5%human plasma and 100 IU/mL of IL-2. 100 μL target cells were co-culturedwith 100 μL effector cells at various E/T ratios for 2 h at 37° C. Thensamples were washed once in FACS buffer. Spontaneous target-cell lysiswas determined in samples only containing labeled target cells. 250 μLpropidium iodide solution (1 μg/mL) were added to the samples shortlybefore measurement. Cells were analyzed in a FACSCanto flow cytometer(BD Biosciences). The percentage of dead target cells was determinedusing FACSDiVa software (BD Biosciences).

REFERENCES

-   Uherek C, Groner B, Wels W. Chimeric antigen receptors for the    retargeting of cytotoxic effector cells. J. Hematother. Stem Cell    Res. 10: 523-543, 2001.-   Uherek C, Tonn T, Uherek B, Becker S, Schnierle B, Klingemann H G,    Wels W. Retargeting of natural killer-cell cytolytic activity to    ErbB2 expressing cancer cells results in efficient and selective    tumor cell destruction. Blood 100: 1265-1273, 2002.-   Fitzer-Attas C J, Schindler D G, Waks T, Eshhar Z. Harnessing Syk    family tyrosine kinases as signaling domains for chimeric single    chain of the variable domain receptors: optimal design for T cell    activation. J Immunol. 160(1):145-154, 1998.-   Müller T, Uherek C, Maki G, Chow K U, Schimpf A, Klingemann H G,    Tonn T, Wels W S. Expression of a CD20-specific chimeric antigen    receptor enhances cytotoxic activity of NK cells and overcomes    NK-resistance of lymphoma and leukemia cells. Cancer Immunol.    Immunother. 57: 411-423, 2008.-   Morgan R A, Dudley M E, Wunderlich J R, Hughes M S, Yang J C, Sherry    R M, Royal R E, Topalian S L, Kammula U S, Restifo N P, Zheng Z,    Nahvi A, de Vries C R, Rogers-Freezer L J, Mavroukakis S A,    Rosenberg S A. Cancer regression in patients after transfer of    genetically engineered lymphocytes. Science. 2006 Oct. 6;    314(5796):126-9.-   Rosenberg S A, Restifo N P, Yang J C, Morgan R A, Dudley M E.    Adoptive cell transfer: a clinical path to effective cancer    immunotherapy. Nat Rev Cancer. 2008 April; 8(4):299-308.

We claim:
 1. A protein comprising (i) a signal peptide; (ii) a targetspecific recognition domain; (iii) a linker region, connecting thetarget specific recognition domain and an effector domain, wherein thelinker region does not contain cysteine residue(s) and consists of: a)the amino acid sequence of SEQ ID NO: 2, or b) an amino acid sequencethat differs in one, two or three amino acid residues from the aminoacid sequence of SEQ ID NO: 2 under the proviso that amino acid residue48 is not a cysteine and is a serine; and (v) the effector domaincomprises: a) a transmembrane domain and an intracellular domain ofhuman costimulatory CD28 receptor fused to an intracellular domain of azeta-chain of a human CD3 complex of a T-cell receptor, or b) azeta-chain of a human CD3 complex of a T-cell receptor with the aminoacid sequence encoded by the nucleic acid sequence of SEQ ID NO:
 9. 2.The protein according to claim 1, wherein the target specificrecognition domain (ii) binds to a tumor-associated surface antigen, alineage-specific or tissue-specific surface antigen or a virus-specificsurface antigen.
 3. The protein according to claim 1, wherein the targetspecific recognition domain (ii) is an antibody or an antigen bindingfragment a single chain Fv (scFv) fragment, Fab fragment, a diabody, ora heavy chain variable domain and a light chain variable domain.
 4. Theprotein according to claim 1, comprising: a) an amino acid sequence ofSEQ ID NO: 7, b) an amino acid sequence encoded by the nucleic acidsequence of SEQ ID NO: 12, c) an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 7 under theproviso that amino acid residue no. 308 is not a cysteine and is aserine, d) an amino acid sequence of SEQ ID NO: 6, e) an amino acidsequence encoded by the nucleic acid sequence of SEQ ID NO: 11, or f) anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 6 under the proviso that amino acid residueno. 308 is not a cysteine and is a serine.
 5. The protein according toclaim 1, comprising: a) an amino acid sequence of SEQ ID NO: 5, b) anamino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:10, c) an amino acid sequence comprising the amino acid residues 29 to163 of SEQ ID NO: 3, or d) an amino acid sequence encoded by the nucleicacid sequence of SEQ ID NO:
 9. 6. The protein according to claim 1,comprising: a) the amino acid sequence of SEQ ID NO: 2, b) an amino acidsequence encoded by the nucleic acid sequence of SEQ ID NO: 8, or c) anamino acid sequence that differs in one, two or three amino acidresidues from the amino acid sequence of SEQ ID NO: 2 under the provisothat amino acid residue 48 is not a cysteine and is a serine.
 7. Apharmaceutical composition comprising the protein of claim 1 and apharmaceutical carrier.